Role of vitamin B12 in
human metabolic processes

Although the nutritional literature still uses the term
vitamin B12,a more specific name for vitamin B12
is cobalamin. Vitamin B12 is the largest of the B complex
vitamins, with a molecular weight of over 1000. It consists of a corrin ring
made up of four pyrroles with cobalt at the center of the ring (1,
2).

There are several vitamin B12-dependent enzymes in
bacteria and algae, but no species of plants have the enzymes necessary for
vitamin B12 synthesis. This fact has significant implications for the
dietary sources and availability of vitamin B12. In mammalian cells
there are only two vitamin B12-dependent enzymes (3). One of
these enzymes, methionine synthase, uses the chemical form of the vitamin which
has a methyl group attached to the cobalt and is called methylcobalamin (see
Figure 7 in Chapter 4.). The other enzyme, methylmalonyl CoA
mutase, uses vitamin B12 with a 5-adeoxyadenosyl moiety
attached to the cobalt and is called 5-deoxyaldenosylcobalamin, or
coenzyme B12. In nature there are two other forms of vitamin
B12: hydroxycobalamin and aquacobalamin, where hydroxyl and water
groups, respectively, are attached to the cobalt. The synthetic form of vitamin
B12 found in supplements and fortified foods is cyanocobalamin, which
has cyanide attached to the cobalt. These three forms of B12 are
enzymatically activated to the methyl- or deoxyadenosylcobalamins in all
mammalian cells.

Dietary sources and
availability

Most microorganisms, including bacteria and algae, synthesise
vitamin B12, and they constitute the only source of the vitamin
(4). The vitamin B12 synthesised in microorganisms enters the
human food chain through incorporation into food of animal origin. In many
animals gastrointestinal fermentation supports the growth of these vitamin
B12-synthesising microorganisms, and subsequently the vitamin is
absorbed and incorporated into the animal tissues. This is particularly true for
the liver, where vitamin B12 is stored in large concentrations.
Products from these herbivorous animals, such as milk, meat, and eggs,
constitute important dietary sources of the vitamin unless the animal is
subsisting in one of the many regions known to be geochemically deficient in
cobalt (5). Milk from cows and humans contains binders with very high
affinity for vitamin B12, whether they hinder or promote intestinal
absorption is not entirely clear. Omnivores and carnivores, including humans,
derive dietary vitamin B12 from animal tissues or products (i.e.,
milk, butter, cheese, eggs, meat, poultry, etc.). It appears that no significant
amount of the required vitamin B12 by humans is derived from
microflora, although vegetable fermentation preparations have also been reported
as being possible sources of vitamin B12 (6).

Absorption

The absorption of vitamin B12 in humans is complex
(1, 2). Vitamin B12 in food is bound to proteins and is
released from the proteins by the action of a high concentration of hydrochloric
acid present in the stomach. This process results in the free form of the
vitamin, which is immediately bound to a mixture of glycoproteins secreted by
the stomach and salivary glands. These glycoproteins, called R-binders (or
haptocorrins), protect vitamin B12 from chemical denaturation in the
stomach. The stomachs parietal cells, which secrete hydrochloric acid,
also secrete a glycoprotein called intrinsic factor. Intrinsic factor binds
vitamin B12 and ultimately enables its active absorption. Although
the formation of the vitamin B12 - intrinsic factor complex was
initially thought to happen in the stomach, it is now clear that this is not the
case. At an acidic pH the affinity of the intrinsic factor for vitamin
B12 is low whereas its affinity for the R-binders is high. When the
contents of the stomach enter the duodenum, the R-binders become partly digested
by the pancreatic proteases, which causes them to release their vitamin
B12. Because the pH in the duodenum is more neutral than that in the
stomach, the intrinsic factor has a high binding affinity to vitamin
B12, and it quickly binds the vitamin as it is released from the
R-binders. The vitamin B12-intrinsic factor complex then proceeds to
the lower end of the small intestine, where it is absorbed by phagocytosis by
specific ileal receptors (1, 2).

Populations at risk for and
consequences of vitamin B12 deficiency

Vegetarians

Because plants do not synthesise vitamin B12,
individuals who consume diets completely free of animal products (vegan diets)
are at risk of vitamin B12 deficiency. This is not true of
lacto-ovo-vegetarians, who consume the vitamin in eggs, milk, and other dairy
products.

Pernicious anaemia

Malabsorption of vitamin B12 can occur at several
points during digestion (1, 4). By far the most important condition
resulting in vitamin B12 malabsorption is the auto-immune disease
called pernicious anaemia (PA). In most cases of PA, antibodies are produced
against the parietal cells causing them to atrophy, lose their ability to
produce intrinsic factor, and secrete hydrochloric acid. In some forms of PA the
parietal cells remain intact but auto-antiobodies are produced against the
intrinsic factor itself and attach to it, thus preventing it from binding
vitamin B12. In another less common form of PA, the antibodies allow
vitamin B12 to bind to the intrinsic factor but prevent the
absorption of the intrinsic factor-vitamin B12 complex by the ileal
receptors. As is the case with most auto-immune diseases, the incidence of PA
increases markedly with age. In most ethnic groups it is virtually unknown to
occur before the age of 50, with a progressive rise in incidence thereafter
(4). However, African American populations are known to have an earlier
age of presentation (4). In addition to causing malabsorption of dietary
vitamin B12, PA also results in an inability to reabsorb the vitamin
B12 which is secreted in the bile. Biliary secretion of vitamin
B12 is estimated to be between 0.3 and 0.5 µg/day. Interruption
of this so-called enterohepatic circulation of vitamin B12 causes the
body to go into a significant negative balance for the vitamin. Although the
body typically has sufficient vitamin B12 stores to last 3-5 years,
once PA has been established the lack of absorption of new vitamin
B12 is compounded by the loss of the vitamin because of negative
balance. When the stores have been depleted, the final stages of deficiency are
often quite rapid, resulting in death in a period of months if left
untreated.

Atrophic gastritis

Historically, PA was considered to be the major cause of
vitamin B12 deficiency, but it was a fairly rare condition, perhaps
affecting 1 percent to a few percent of elderly populations. More recently it
has been suggested that a far more common problem is that of hypochlorhydria
associated with atrophic gastritis, where there is a progressive reduction with
age of the ability of the parietal cells to secrete hydrochloric acid
(7). It is claimed that perhaps up to one-quarter of elderly subjects
could have various degrees of hypochlorhydria as a result of atrophic gastritis.
It has also been suggested that bacterial overgrowth in the stomach and
intestine in individuals suffering from atrophic gastritis may also reduce
vitamin B12 absorption. This absence of acid is postulated to prevent
the release of protein-bound vitamin B12 contained in food but not to
interfere with the absorption of the free vitamin B12 found in
fortified foods or supplements. Atrophic gastritis does not prevent the
reabsorption of bilary vitamin B12 and therefore does not result in
the negative balance seen in individuals with PA. However, it is agreed that
with time, a reduction in the amount of vitamin B12 absorbed from the
diet will eventually deplete even the usually adequate vitamin B12
stores, resulting in overt deficiency.

When considering recommended nutrient intakes (RNIs) for
vitamin B12 for the elderly, it is important to take into account the
absorption of vitamin B12 from sources such as fortified foods or
supplements as compared with dietary vitamin B12. In the latter
instances, it is clear that absorption of intakes of less than 1.5-2.0
µg/day is complete - that is, for intakes of less than 1.5-2.0 µg of
free vitamin B12, the intrinsic factor - mediated system absorbs all
of that amount. It is probable that this is also true of vitamin B12
in fortified foods, althoughthis has not specifically been
examined. However, absorption of food-bound vitamin B12 has been
reported to vary from 9 percent to 60 percent depending on the study and the
source of the vitamin, which is perhaps related to its incomplete release from
food (8). This has led many to estimate absorption as being up to 50 percent to
correct for bio-availability of absorption from food.

Vitamin B12 interaction
with folate or folic acid

One of the vitamin B12 - dependent enzymes,
methionine synthase, functions in one of the two folate cycles (see
Chapter 4) - the methylation cycle. This cycle is necessary to
maintain availability of the methyl donor S-adenosylmethionine;
interruption reduces the wide range of methylated products. One such important
methylation is that of myelin basic protein. Reductions in the level of
S-adenosylmethionine seen in PA and other causes of vitamin
B12 deficiency produce demyelination of the peripheral nerves and the
spinal column, called sub-acute combined degeneration (1, 2). This
neuropathy is one of the main presenting conditions in PA. The other principal
presenting condition in PA is a megaloblastic anaemia morphologically identical
to that seen in folate deficiency. Disruption of the methylation cycle should
cause a lack of DNA biosynthesis and anaemia.

The methyl trap hypothesis is based on the fact that once the
cofactor 5,10-methylenetetrahydrofolate is reduced by its reductase to form
5-methyltetrahydrofolate, the reverse reaction cannot occur. This suggests that
the only way for the methyltetrahydrofolate to be recycled to tetrahydrofolate,
and thus to participate in DNA biosynthesis and cell division, is through the
vitamin B12 - dependent enzyme methionine synthase. When the activity
of this synthase is compromised, as it would be in PA, the cellular folate will
become progressively trapped as 5-methyltetrahydrofolate. This will result in a
cellular pseudo folate deficiency where despite adequate amounts of folate an
anaemia will develop that is identical to that seen in true folate deficiency.
Clinical symptoms of PA, therefore, include neuropathy, anaemia, or both.
Treatment with vitamin B12, if given intramuscularly, will reactivate
methionine synthase, allowing myelination to restart. The trapped folate will be
released and DNA synthesis and generation of red cells will cure the anaemia.
Treatment with high concentrations of folic acid will treat the anaemia but not
the neuropathy of PA. It should be stressed that the so-called masking of the
anaemia of PA is generally agreed not to occur at concentrations of folate found
in food or at intakes of the synthetic form of folic acid found at usual RNI
levels of 200 or 400 µg/day (1). However, there is some evidence
that amounts less than 400 µg may cause a haematologic response and thus
potentially treat the anaemia (9). The masking of the anaemia definitely
occurs at high concentrations of folic acid (>1000 µg/day). This becomes
a concern when considering fortification with synthetic folic acid of a dietary
staple such as flour (see Chapter 4).

In humans the vitamin B12 - dependent enzyme
methylmalonyl coenzyme A (CoA) mutase functions in the metabolism of propionate
and certain of the amino acids, converting them into succinyl CoA, and in their
subsequent metabolism via the citric acid cycle. It is clear that in vitamin
B12 deficiency the activity of the mutase is compromised, resulting
in high plasma or urine concentrations of methylmalonic acid (MMA), a
degradation product of methylmalonyl CoA. In adults this mutase does not appear
to have any vital function, but it clearly has an important role during
embryonic life and in early development. Children deficient in this enzyme,
through rare genetic mutations, suffer from mental retardation and other
developmental defects.

Assessment of vitamin B12
status

Traditionally it was thought that low vitamin B12
status was accompanied by a low serum or plasma vitamin B12 level
(4). Recently this has been challenged by Lindenbaum et
al.(10), who suggested that a proportion of people with normal
vitamin B12 levels are in fact vitamin B12 deficient. They
also suggested that elevation of plasma homo-cysteine and plasma MMA are more
sensitive indicators of vitamin B12 status. Although plasma
homo-cysteine may also be elevated because of folate or vitamin B6
deficiency, elevation of MMA apparently always occurs with poor vitamin
B12 status. There may be other reasons why MMA is elevated, such as
renal insufficiency, so the elevation of itself is not diagnostic. Many would
feel that low or decreased plasma vitamin B12 levels should be the
first indication of poor status and that this could be confirmed by an elevated
MMA if this assay was available.

Evidence on which to base a
recommended intake

Recommendations for nutrient intake

The Food and Nutrition Board of the National Academy of
Sciences (NAS) Institute of Medicine (8) has recently exhaustively
reviewed the evidence of intake, status, and health for all age groups and
during pregnancy and lactation. This review has lead to calculations of what
they have called an estimated average requirement (EAR). The EAR is defined by
NAS as the daily intake value that is estimated to meet the requirement,
as defined by the specific indicator of adequacy, in half of the individuals in
a life-stage or gender group (8). They have estimated the
recommended dietary allowances to be this figure plus 2 standard deviations
(SDs). Some members of the Food and Agriculture Organization of the United
Nations and World Health Organization (FAO/WHO) Expert Consultation were
involved in the preparation and review of the NAS recommendations and judge them
to have been the best estimates based on available scientific literature. The
FAO/WHO expert group felt it appropriate to adopt the same approach used by the
NAS in deriving the RNIs. Therefore the RNIs suggested in Table 14
are based on the NAS EARs plus 2 SDs.

Adults

Several lines of evidence point to an adult average
requirement of about 2.0 µg/day. The amount of intramuscular vitamin
B12 needed to maintain remission in people with PA suggests a
requirement of about 1.5 µg/day (10), but they would also be losing
0.3-0.5µg/day through interruption of their enterohepatic circulation,
which is not typical. This might suggest a requirement of 0.7-1.0 µg/day.
Because vitamin B12 is not completely absorbed from food, an
adjustment of 50 percent has to be added giving a range of 1.4-2.0 µg/day
(4). Therapeutic response to ingested food vitamin B12
suggests a minimum requirement of something less than 1.0 µg/day
(8). Diets containing 1.8 µg/day seemed to maintain adequate status
but lower intakes showed some signs of deficiency. (8). Dietary intakes
of less than 1.5 µg/day were reported to be inadequate in some subjects
(11).

In summary, the average requirement could be said to be 2
µg/day (8). The variability of the requirements for vitamin
B12 is accounted for by adding two SDs, that is, 2.4 µg/day as
an RNI for adults, including the elderly.

Table 14

Estimated average requirement (EAR) and recommended
nutrient intake (RNI) for vitamin B12, by age group

Adapted from the US National Academy of Sciences
(8).

Group

EAR µg/day

RNI µg/day

Infants and children

0-6 months

0.32

0.4

7-12 months

0.32

0.5

1-3 years

0.7

0.9

4-6 years

1.0

1.2

7-9 years

1.5

1.8

Adolescents, 10-18 years

2.0

2.4

Adults

19-65 years

2.0

2.4

65+ years

2.0

2.4

Pregnancy

2.2

2.6

Lactation

2.4

2.8

Children

The Food and Nutrition Board of the NAS Institute of Medicine
(8) suggested the same intakes for adolescents with progressive reduction
of intake for younger groups.

Pregnancy

The previous FAO/WHO (12) Expert Consultation suggested that
0.1-0.2 µg/day of vitamin B12 is transferred to the foetus
(13) during the last two trimesters of pregnancy. On the basis of on
foetal liver content from post-mortem samples (14, 15, 16), there
is further evidence that the foetus accumulates on average 0.1-0.2 µg/day
during pregnancies of women with diets which have adequate vitamin
B12. It has been reported that children born to vegetarians or other
women with a low vitamin B12 intake subsequently develop signs of
clinical vitamin B12 deficiency such as neuropathy (17).
Therefore, when calculating the EAR for pregnant women, 0.2 µg/day of
vitamin B12 is added to the EAR for adults to result in an EAR of 2.2
µg/day and an RNI of 2.6 µg/day during
pregnancy.

Lactation

It is estimated that 0.4 µg/day of vitamin B12
is found in the human milkof women with adequate vitamin B12
status (8). Therefore an extra 0.4 µg/day of vitamin B12
is needed during lactation in addition to the normal adult requirement of
2.0 µg/day, giving a total EAR of 2.4 µg/day during lactation and an
RNI of 2.8 µg/day.

Infants

As with other nutrients, the principal way to determine
requirements of infants is to examine the levels in milk from mothers on
adequate diets. There is a wide difference in the vitamin B12 values
reported in human milk because of differences in methodology. The previous
FAO/WHO report (12) used milk vitamin B12 values of normal women of
about 0.4 µg/l. For an average milk production of 0.75 l/day, the vitamin
B12 intake by infants would be 0.3 µg/day (18). Other
studies have reported ranges of vitamin B12 in human milk to be
0.4-0.8 µg/L (17, 19, 20, 21, 22). Although daily intakes ranging
from 0.02 to 0.05 µg/day have been found to prevent deficiency (23,
24), these intakes are totally inadequate for long-term health. An EAR of
0.3-0.6 µg/day would result in an RNI of 0.36-0.72 µg/d. It might be
prudent to use the lower figure of 0.4 µg/day for the first 6 months of
pregnancy and 0.7 µg/day for last trimester.

Upper limits

The absorption of vitamin B12 mediated by intrinsic
factor is limited to 1.5-2.0 µg per meal because of the limited capacity of
the receptors. In addition, between 1 percent and 3 percent of any particular
oral administration of vitamin B12 is absorbed by passive diffusion.
Thus, if 1000 µg vitamin B12 (sometimes used to treat those with
PA) is taken orally, the amount absorbed would be 2.0 µg by active
absorption plus about 30 µg by passive diffusion. This amount has never
been reported to have any side effects (8). Similar large amounts have
been used in some preparations of nutritional supplements without apparent ill
effects. However, there are no established benefits for such amounts. Such high
intakes thus represent no benefit in those without malabsorption and should
probably be avoided.

Future research

Because they do not consume any animal products, vegans are at
risk of vitamin B12 deficiency. It is generally agreed that in some
communities the only source of vitamin B12 is from contamination of
food by microorganisms. When vegans move to countries where standards of hygiene
are more stringent, there is good evidence that risk of vitamin B12
deficiency increases in adults and, particularly, in children born to and
breast-fed by women who are strict vegans.

As standards of hygiene improve in developing countries, there is a concern
that the prevalence of vitamin B12 deficiency might increase. This
should be ascertained by estimating plasma vitamin B12 levels,
preferably in conjunction with plasma MMA levels in representative adult populations
and in infants.

The contribution which fermented vegetable foods make to vitamin B12
status of vegan communities should be investigated.

The prevalence of atrophic gastritis should be investigated in developing
countries.